Prevalence and incidence of anal high‐grade squamous intraepithelial lesions in a cohort of cisgender men and transgender women who have sex with men diagnosed and treated during acute HIV acquisition in Bangkok, Thailand

Abstract Introduction Men who have sex with men (MSM), especially those living with HIV, are at an increased risk of anal cancer. The prevalence and incidence of its precursor, anal high‐grade squamous intraepithelial lesions (HSILs), among MSM who started antiretroviral therapy during acute HIV acquisition are yet to be explored. Methods Participants in an acute HIV acquisition cohort in Bangkok, Thailand, who agreed to take part in this study, were enrolled. All participants were diagnosed and started antiretroviral therapy during acute HIV acquisition. Human papillomavirus (HPV) genotyping and high‐resolution anoscopy, followed by anal biopsy as indicated, were done at baseline and 6‐monthly visits. Results A total of 89 MSM and four transgender women were included in the analyses. Median age at enrolment was 26 years. Baseline prevalence of histologic anal HSIL was 11.8%. With a total of 147.0 person‐years of follow‐up, the incidence of initial histologic anal HSIL was 19.7 per 100 person‐years. Factors associated with incident anal HSIL were anal HPV 16 (adjusted hazards ratio [aHR] 4.33, 95% CI 1.03–18.18), anal HPV 18/45 (aHR 6.82, 95% CI 1.57–29.51), other anal high‐risk HPV (aHR 4.23, 95% CI 1.27–14.14), syphilis infection (aHR 4.67, 95% CI 1.10–19.90) and CD4 count <350 cells/mm3 (aHR 3.09, 95% CI 1.28–7.48). Conclusions With antiretroviral therapy initiation during acute HIV acquisition, we found the prevalence of anal HSIL among cisgender men and transgender women who have sex with men to be similar to those without HIV. Subsequent anal HSIL incidence, although lower than that of those with chronic HIV acquisition, was still higher than that of those without HIV. Screening for and management of anal HSIL should be a crucial part of long‐term HIV care for all MSM.


I N T R O D U C T I O N
Anal cancer is a rising concern for people living with HIV.The incidence of anal cancer among men who have sex with men (MSM) with HIV was 85 per 100,000 person-years, a rate five times higher than MSM without HIV [1].It is not mitigated by the use of antiretroviral therapy (ART) [2,3].Human papillomavirus (HPV) infection is the major cause of anal cancer [4,5], with HPV 16 responsible for 70% of cases in MSM with HIV [4].
Anal high-grade squamous intraepithelial lesions (HSILs) are the known precursors of anal cancer [6,7].Persistent HPV 16 is associated with persistent anal HSIL [8] and the greatest risk of anal cancer [8,9].Previous studies have shown differences in the prevalence and incidence of anal HSIL between people living with HIV compared to those without HIV [8][9][10][11][12].The pooled prevalence of anal HSIL among MSM with HIV from a recent systematic review was 22.4%, twice as high as among MSM without HIV [12].The incidence of anal HSIL among people living with HIV is also generally higher than that of people without HIV (10.5-31.3 vs. 4.9-10.1 per 100 person-years) [8,10,11,13,14].A recent study demonstrates that screening for and treating anal HSIL among MSM with HIV effectively prevents progression to anal cancer in almost 60% of cases [15].
Early ART initiation has been shown to preserve the immune system [16][17][18], but the prevalence and incidence of anal HSIL among those who initiate ART during acute HIV acquisition, before permanent immunologic damage, remains unknown.Therefore, the aim of this study is to describe the prevalence, incidence and associated factors related to anal HSIL progression among cisgender men and transgender women (TGW) living with HIV who began ART during acute HIV acquisition.

M E T H O D S 2.1 Study design and population
This is a prospective observational cohort of cisgender men and TGW who have sex with men who initiated ART at the time of acute HIV acquisition diagnosis in the SEARCH010/RV254 cohort (ClinicalTrials.govIdentifier NCT00796146) in Bangkok, Thailand.Acute HIV acquisition was defined as Fiebig stages 1-5 [19] HIV acquisition.All participants were offered immediate ART.They were examined at baseline to determine the prevalence of anal HSIL and HPV infection within 1 week after ART initiation.They were then followed up every 6 months for the occurrence of anal HSIL over time.This study analysed data collected from participants enrolled from May 2017 to June 2020.Data from follow-up visits until June 2021 were included in the analysis.Data obtained at the baseline visit included demographic data, circumcision history, age of sexual debut, sexual behaviour, history of condom use, sexually transmitted infections, smoking, alcohol drinking and amphetamine-type stimulant use.Laboratory tests, including sexually transmitted infection testing, CD4 count and plasma HIV-1 RNA, were obtained from the SEARCH010/RV254 study database.Digital anorectal examination, anal cytology, HPV genotyping and high-resolution anoscopy (HRA), followed by anal biopsy as indicated, were performed at the baseline and subsequent visits.This study analysed data collected from participants enrolled between May 2017 and June 2020.Data from follow-up visits until June 2021 were included in the analysis.
All participants provided informed consent.The study was approved by the institutional review board of Chulalongkorn University in Bangkok, Thailand (IRB No. 706/59) and registered at ClinicalTrials.gov(Identifier NCT03032575).

Anal cytology, HRA and biopsy
Anal specimens were collected prior to HRA at each visit.A moistened, non-lubricated polyester-tipped swab was inserted 5-7 centimetres into the anal canal and slowly removed with a twirling motion and gentle pressure applied to the walls of the anal canal to maximize cellular yield.The swab was placed in a liquid-based cytology fluid (ThinPrep®, Cytyc Corporation, Boxborough, MA, USA) for transport and storage until processing.HRA was performed at baseline and every 6 months by the same study physician (NT).Acetic acid solution and Lugol's solution were used to aid in the visualization of abnormal anal tissue.Anal biopsy was performed on suspicious lesions and processed and assessed by the pathology department of King Chulalongkorn Memorial Hospital.Cytology and histology results were reported in accordance with the terminology of the Lower Anogenital Squamous Terminology (LAST) Project [20].Discrepancies were resolved by re-evaluation, discussion and concurrence by at least two2 pathologists.Participants who had anal HSIL at their last study visits were offered treatment with infrared coagulation free of charge following the local standard of care.

HPV genotyping
HPV genotyping was performed on liquid-based anal cytology fluid collected at baseline and every 6 months and stored at −80°C

Laboratory testing
Testing for sexually transmitted infections, CD4 count and plasma HIV-

Statistical analysis
Categorical variables are presented as counts and percentages.Continuous variables are presented as medians and interquartile ranges (IQRs).The prevalence of anal HSIL was calculated together with Wilson binomial 95% confidence intervals (CIs).Formal comparisons of continuous and categorical characteristics of participants by the HSIL group were made with a Wilcoxon rank-sum test and Fisher's exact test, respectively.The incidence of anal HSIL was calculated per person-time at risk.Person-time was calculated using the actual participant visit dates.Only the first diagnosed HSIL per participant during the study period was counted as an incident case for calculating HSIL incidence.Histologic anal HSIL was defined as either histologic anal intraepithelial neoplasia (AIN) 2 with positive p16 immunohistochemistry staining or AIN 3. A composite anal HSIL endpoint was defined as a histologic HSIL and/or cytologic HSIL or atypical squamous cells, cannot exclude HSIL.The prevalence and incidence of anal HSIL were calculated.High-risk anal HPV infection was categorized into three groups: 16, 18/45 and other, based on the categorization limit of the Cepheid Xpert HPV assay (Gen-eXpert; Cepheid, Sunnyvale, CA) that was used in recent visits by the cohort.The current study does not contain data generated using this assay.The CD4 count was categorized using a cutoff of 350 cells/mm 3 to represent advanced HIV acquisition [21].The plasma HIV RNA was categorized based on the median as a cutoff.Factors associated with the prevalence of histologic anal HSIL were assessed using a logistic regression.A threshold of a p-value of <0.2 was used to select variables to adjust for in a multivariable model.Factors associated with the incidence of histologic anal HSIL were assessed using the Kaplan−Meier method and Cox-proportional hazards regression.A multivariable model was built using variables with p<0.

Baseline characteristics of participants
A total of 96 cisgender men and TGW who have sex with men with acute HIV acquisition underwent screening from May 2017 to June 2020 (Table 1 S1).

Participant retention and follow-up data
Follow-up duration from the baseline visit to the last visit ranges from 0 to 46 months (median 33).Cohort retention flowchart is shown in Figure S1.By week 24 of follow-up, the majority of participants' CD4 count became ≥350 cells/mm 3 , and all of them were virally suppressed (plasma HIV RNA <1000 copies/ml).Progression of CD4 count and plasma HIV RNA throughout the follow-up period can be seen in Table S2.

Prevalence and incidence of anal HSIL
Histologic anal HSIL was identified in 11/93 individuals (11.8%) at baseline (Table 2).The prevalence of composite anal HSIL was the same.In the multivariable analysis, only plasma HIV RNA ≥6 log 10 copies/ml was associated with prevalent anal HSIL adjusted for age (adjusted odds ratio 8.50, 95% CI 1.03-69.93;Table 3).
Of the 82 individuals who did not have anal HSIL at baseline, 80 were followed up during the study period.The time to first incidence of anal HSIL ranged from 5.2 to 44.6 months (median 22.1).The number of person-years at risk when considering the histologic end point totalled 147.0.During this follow-up time, 29 out of 80 individuals developed incident histologic anal HSIL.This translates to a 19.7 per 100 personyears incidence rate of initial histologic anal HSIL (95% CI 13.7-28.4;Table 2).
Overall, the cumulative probability of incident anal HSIL by month 24 after the diagnosis of acute HIV acquisition was 35.0%(95% CI 25.0%-47.4%),and at month 44 was 48.8% (95% CI 34.0%-66.1%).As shown in Figure 1, there was a significant difference in the probability of initial anal HSIL over time between individuals with and without baseline anal high-risk HPV infection (p = 0.004).Among those with baseline anal high-risk HPV infection, the median (25th percentile) time from acute HIV acquisition to incident histologic anal HSIL was 27.2 (11.0) months in those with baseline anal highrisk HPV infection, while among those without baseline highrisk HPV infection, incident HSIL did not reach 50%, but the 25th percentile was 38.3 months.We found no difference in the probability of anal HSIL over time between individuals infected with anal HPV 16, 18 or 45, and other high-risk HPV genotypes (log-rank p = 0.833).

D I S C U S S I O N
We demonstrated for the first time the prevalence and incidence of anal HSIL among cisgender men and TGW who have sex with men who were diagnosed and treated during acute HIV acquisition.The anal HSIL prevalence of 11.8% at HIV acquisition and diagnosis was comparable to that of 11.4% in HIV-negative Thai MSM [10] and a pooled prevalence of 11.3% in a systematic global review of HIV-negative MSM [12].However, we observed that the incidence of initial anal HSIL among people who initiated ART during acute HIV acquisition was about midway between those previously reported in HIV-negative and chronic HIV cohorts.The incidence of initial histologic anal HSIL in the current acute HIV cohort was    19.7 per 100 person-years, compared with 7.3 and 31.3 per 100 person-years in Thai MSM without and with HIV, respectively [10].This finding supports previous evidence that early detection and treatment of HIV in the acute stage of acquisition prevents the immunological dysfunction seen in individuals with chronically acquired HIV [16-18, 22, 23], potentially protecting against HSIL through the prevention or early resolution of HPV acquisition.However, the incidence of anal HSIL as seen in our study was still higher than that of HIV-negative individuals.
Our study suggests that using histologic anal HSIL is sufficient as a diagnostic endpoint, as the incremental yield of adding cytologic methods resulted in only one additional diagnosis of HSIL, raising the proportion of the longitudinal cohort diagnosed with HSIL from 29/80 (36.3%) to 30/80 (37.5%).However, this conclusion may not be readily generalizable, as the histologic detection of anal HSIL is very much operator dependent.The ability to detect histologic anal HSIL increases with the experience of the anoscopist [24,25].Our anoscopist (NT) had 8 years of experience with HRA at study initiation.In contrast to our findings, another study recommended the use of combined cytologic and histologic anal HSIL endpoints, instead of cytologic or histologic endpoints alone, to capture the majority of anal HSIL [26].
In our cohort, incident histologic anal HSIL was associated with baseline syphilis status, anal high-risk HPV infection and CD4 count.Infection with anal high-risk HPV genotypes is indeed the main cause of anal HSIL [4,5,8].Our study demonstrates that anal high-risk HPV acquisition is associated with a four-to seven-fold increase in the relative incidence of anal HSIL.Thus, HPV vaccines should be given to MSM to prevent anal HPV acquisition [27], preferably before their first sexual encounter.In Thailand, the barrier to receiving HPV vaccines for men is the high price and lack of coverage through the universal health insurance programme.Furthermore, individuals with a lower CD4 count at acute HIV diagnosis were also more likely to develop anal HSIL than those with a higher CD4 count.This indicates that the magnitude of the decline in CD4 count during the acute stage of HIV acquisition, although temporary, has long-term consequences and is a predictor of future progression to anal HSIL.
The current study has some limitations.Firstly, almost all the study participants were male-at-birth Thai people who have had sex with men.Therefore, the findings might not be applicable to other ethnic groups or genders.Secondly, the number of participants was small, since it is uncommon to detect acute HIV acquisition and difficult to recruit people into a study that involves repeated anoscopy and anal biopsy.However, this is the largest cohort to date to study anal HSIL prevalence and incidence among individuals diagnosed and treated during acute HIV acquisition.Thirdly, as discussed earlier, the histologic diagnosis of anal HSIL depends highly on the experience of the operators; hence, the results may vary with other anoscopists.Finally, some variables, such as the number of sexual partners and years since first sex, were based solely on self-report and were vulnerable to recall bias.

Figure 1 .
Figure 1.Kaplan−Meier curve of the cumulative probability of initial anal HSIL among participants free of prevalent anal HSIL, stratified by anal high-risk HPV infection status.Note: Median survival of anal HSIL among any baseline anal high-risk HPV infection was 27.2 months.

Table 1 . (Continued)
a Not including missing or invalid values in the analysis.b Two participants positive for both HPV 16 and 45.

Table 3 . (Continued)
Abbreviations: aOR, adjusted odds ratio; ATS, amphetamine-type stimulant; CI, confidence interval; HPV, human papillomavirus; OR, odds ratio.a Two participants positive for both HPV 16 and 45 were grouped in the HPV 16 category.